Synthetic Probes of Protein Prenylation: Covalent modification by isoprenoid lipids (prenylation) is a critical post-translational event for many proteins involved in cellular signaling. The discovery that the members of the Ras family of protooncogenes are modified by the farnesyl isoprenoid (farnesylation), and that prenylation is required for the oncogenic forms of these proteins to express their transforming potential, has led to intense investigation of protein-farnesyltransferase (FTase) inhibitors (FTIs) as promising cancer chemotherapeutic agents. However, recent developments have made it clear that the mechanism of FTI action is unexpectedly complex, although it involves inhibition of FTase. In addition, the contribution of isoprenoid lipids to the overall biology of Ras is incompletely understood. Therefore, a key to applying FTase-based pharmacological intervention is a thorough understanding of the in vivo farnesylation pathways. Knowledge of the substrate specifities for FTase, and the cellular function of the prenyl moiety are critical to improving the design of future FTIs. The central hypothesis of this study is that the prenyl group plays an active role in directing both post- translational processing and cellular membrane localization of prenylated proteins. An important corollary to this hypothesis is that modifications to the prenyl structure may lead to significant, biologically relevant effects on the activity of the unnaturally prenylated protein. We have synthesized FPP analogs that are transferred to oncogenic Ras but fail to support transformation. These molecules are leads for a unique class of potential anti-cancer therapeutics we term RFIs (Ras function inhibitors). The specific aims of this project are: 1) specifically substituted unnatural analogs of farnesyl pyrophosphate and the homologous isoprenoid geranylgeranyl pyrophosphate will be synthesized in a combinatorial scheme; 2) these compounds will be screened as substrates or inhibitors of FTase and the closely related enzyme geranylgeranyltransferase I; 3) building upon promising preliminary results in this area, an in vivo isoprenoid structure-function relationship will be established by replacing the H-Ras farnesyl group with a select subset of the analogs available from the studies described in specific aim 1and 2 and analyzing their biological functions following microinjection intoXenopus oocytes. The results of these experiments will provide further insight into the mechanisms of Ras processing and transformation, a greater understanding of the specific functions of the H-Ras farnesyl group in vivo, and fruitful directions for improvements in FTIs as well as novel transferable analogs which might act as RFIs.